The overall goal of this program is to isolate, reconstitute and characterize at the molecular level the glucose carrier of human erythrocyte membranes. More immediate objectives of this proposal include isolation of the glucose-sensitive cytochalasin B binding protein and glucose-specific binding protein, and an unambiguous reconstitution and characterization of the carrier function in vitro in spherical bilayer membranes of a large volume-surface area ratio. The following experiments are proposed: 1. To synthesize photoreactive analogs of cytochalasin B and D-glucose which display appropriately site-specific inhibition of the glucose-sensitive cytochalasin B binding and the carrier-mediated glucose transport. 2. To label the glucose-sensitive cytochalasin B binding protein either with this site-specific, photoreactive (3H) Cytochalasin B analog, or by fixing the binding protein-cytochalasin B complex covalently using cross linking reagents. 3. To identify the glucose carrier (glucose-specific binding) protein either by labeling it with the site-specific, photoreactive (14C1D-glucose analog, or by fixing the glucose carrier-(14C1D-glucose complex covalently using photoreactive cross linking reagents. 4. The isolation and purification of both the glucose-sensitive cytochalasin B binding protein and the D-glucose-specific binding protein will also be approached via selective solubilization and fraction of membrane proteins, followed by affinity chromatographic purification using the immobilized site-specific analogs referred to above. 5. To reconstitute in vitro a glucose carrier activity of high efficiency from these isolates, in a spherical bilayer membrane of a large volume-surface area ratio, and to characterize the reconstituted activity in terms of flux-rate, specificity, kinetics, and pharmacological interactions. 6. To devise a method to prepare a proteoliposome suspension of a uniformally large size, greater than or equal to 1 micron diameter, and to reconstitute the glucose carrier quantitatively in this preparation.